There are several known ways of measuring and obtaining characteristics and various transient-state properties in fluorescent molecules. One way is the so-called fluorescence correlation spectroscopy (FCS) (see J. Widengren and Ü. Mets p. 69-119 in “Single Molecule Detection in Solution—methods and applications” VCH-Wiley Verlag, Berlin, 2002). The use of FCS and related methods have increased dramatically in the last 10 years in academia and among biotechnological and pharmaceutical companies for studies of molecular interactions (see M. Auer, Drug Disc, Today, 1998, 3: 457, C Eggeling, L Brand, D Ullmann and S Jäger, Drug Disc. Today, 1998, 2003 8(14): 632). FCS is based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. The technique can in principle offer information about any molecular dynamic process in the nanosecond time range and longer that manifests itself as a change in fluorescence intensity. The fluorescence fluctuations are analysed in terms of the auto-covariance of the detected fluorescence intensity, normalised by the time-averaged fluorescence intensity squared.
                              G          ⁡                      (            τ            )                          =                                            <                                                F                  ⁡                                      (                    t                    )                                                  ⁢                                  F                  ⁡                                      (                                          t                      +                      τ                                        )                                                              >                                      <              F              ⁢                              >                2                                              =                                                    <                                  δ                  ⁢                                                                          ⁢                                      F                    ⁡                                          (                      0                      )                                                        ⁢                  δ                  ⁢                                                                          ⁢                                      F                    ⁡                                          (                      τ                      )                                                                      >                                            <                F                ⁢                                  >                  2                                                      +            1                                              (                  equation          ⁢                                          ⁢          1                )            
For translational diffusion, the duration of the fluorescence fluctuations reflects the passage times of the molecules, while the relative amplitude of the fluctuations are inversely proportional to the mean number of molecules, N within the detection volume 102 (see FIG. 1). The correlation curve 202 reflects the probability versus time of detecting a fluorescence photon from a molecule, given that a photon was detected at time zero from that molecule (see FIG. 2). Consequently, the decay time of the FCS curve, τD, reflects the passage time. The amplitude is proportional to the relative fluorescence fluctuations and 1/N.
In fluorescent molecules, i.e. fluorophores, emission of a fluorescence photon typically takes place in the subsequent relaxation, k21 302, following excitation of the ground singlet state (S0) to the first excited singlet state (S1) 304. The rate of relaxation from S1 to S0 (k21) is typically of the order 109 s−1. In addition, transition to more long-lived, non- or weakly fluorescent transient states can take place 306, hereafter referred to as the transient state, and among which we take the lowest triplet state as an example to explain the background and illustrate the principles of the invention. Among others, states generated by photo-induced charge transfer or isomerisation in particular belong in the category of such transient states. They can also be monitored by FCS in a principally similar way as the triplet state (see Widengren J, Dapprich J and Rigler R. Chem Physics, 216, 417-426, 1997 and Widengren J. Schwille P. J Phys Chem A. 104(27):6416-6428, 2000).
For the lowest triplet state (T), population takes place by intersystem crossing from S1, typically with a rate (kISC) of the order 106 s−1 (see FIG. 3). The triplet state can be considered as totally non-fluorescent. From the ratio of the rate constants, kISC/k21 it follows that transition to the triplet state takes place in approximately one per thousand of the excitation-emission cycles. However, once populated, the triplet state is quite long-lived (μs-ms). The triplet decay rate, kT, is of the order 103 to 106 s−1. As a consequence, the steady state population of triplet-state fluorophores accumulates strongly at high continuous excitation rates, i.e. when kexc is comparable to k21 so that S1 is significantly populated. Transitions to and from the triplet state generate fluorescence fluctuations, with the fluorophore being fluorescent while in the singlet entity (S0 and S1), but non-fluorescent while residing in the triplet state, T.
In FCS, these fluctuations are superimposed on those due to translational diffusion of the fluorescent molecules into and out of the detection volume 102 (see FIG. 1), and generate a second relaxation process in the correlation curves. In these correlation curves 402, the relative amplitude of this second relaxation process corresponds to the average triplet state population, T, of the fluorophores in the detection volume. The relaxation time, τT, corresponds to the relaxation time of the singlet-triplet transitions (see FIG. 4) (see J Widengren, Ü Mets, R Rigler, J Phys Chem 1995, 99, p. 13368). Following from FIG. 3, both these parameters depend on the excitation rate, k12.
                              T          _                =                                            k              12                        ⁢                          k              23                                                                          k                21                            ⁢                              k                31                                      +                                          k                12                            ⁡                              (                                                      k                    23                                    +                                      k                    31                                                  )                                                                        (                  equation          ⁢                                          ⁢          2                )                                          τ          T                =                  1                                    k              31                        +                                          k                23                            ⁢                                                k                  12                                /                                  (                                                            k                      12                                        +                                          k                      21                                                        )                                                                                        (                  equation          ⁢                                          ⁢          3                )            
Generating FCS curves at different excitation intensities, and determining T and τT, makes it possible to determine all the rate parameters involved in the singlet-triplet transitions. FCS offers several important advantages to flash photolysis or transient absorption techniques for investigations of triplet states. In contrast to these techniques, FCS takes advantage of the inherent thermodynamic fluctuations to follow a kinetic process, without the need of any external synchronisation in the form of a perturbation. Triplet parameters derived from FCS are also well reproducible. In contrast, the reported triplet state rate parameters obtained by transient absorption techniques vary substantially between different reports.
Nonetheless, there are difficulties and disadvantages when using FCS. Although the method is relatively simple and powerful, triplet state monitoring by FCS is also afflicted with certain limitations. One of those is that low molecular numbers have to be recorded in order see the necessary spontaneous fluctuations. Therefore only low sample concentrations (<100 nM) can be monitored. Secondly, highly sensitive detectors with low noise levels are required and the time scale of the triplet fluctuations requires detection and processing of the fluorescence data with high time resolution. Only point detectors can meet these demands on the detection.
Probing the triplet state properties can yield useful molecular information. One use is to probe extent of triplet state induction or quenching. As for fluorescence quenching, these effects are primarily based on collisional interactions between fluorophores and molecules that can induce enhanced transition rates between different states of the fluorophores. In fluorescence quenching an additional non-fluorescent relaxation pathway from S1 to S0 is induced, thereby reducing the fluorescence. The decrease in fluorescence (or the decrease in the fluorescence lifetime of the fluorophore) can then be used to measure the degree of collisional interaction, which for instance can give information on the accessibility of the site of labelling, and how well shielded it is from the surrounding solvent (see FIGS. 5 and 6). The accessibility may change as a consequence of a conformational change or a molecular interaction, and can thus be used to monitor these Triplet states, on the other hand, are relatively long-lived (ms-μs) compared to S1 (ns). Surrounding molecules that can affect the rate of triplet state deactivation, kT, therefore have at least 1000 times as long time to collisionally interact with the triplet state. They can also induce an increased rate of intersystem crossing to T. Triplet state quenching/induction, monitored via triplet state kinetics, can thus give very sensitive information of the immediate environment of the fluorophores, reflecting for example molecular interactions or changes in protein conformations.
Apart from addition of molecules affecting formation or decay of triplet state fluorescent molecules, transitions to and from triplet states can be modified by excitation of S1 and/or T to higher singlet or triplet states. For these higher states, the transition rates between the singlet and triplet entities of the fluorophores are different from those between S1/S0 and T, thereby altering the fraction of fluorophores being in a triplet state. Excitation to higher excited states (by a subsequent radiation field) can be performed at the same time as the excitation responsible for k12, taking S0 to S1 (the k12-excitation), and the optimal excitation wavelength is typically slightly red-shifted compared to that of the k12-excitation. For an example of light-induced triplet quenching/modification by application of an additional excitation field (e.g. a field from an acousto-optical modulator, AOM 1402 in FIG. 14A), generated and monitored in an FCS experiment, see J. Widengren, C. Seidel, Phys Chem Chem Phys, 2000, 2: p. 3435-3441.
The subsequent radiation field can also be applied with some delay in time with respect to the k12-excitation. Moreover, the radiation field can in addition to generating the subsequent excitation to higher excited states also be used to induce stimulated emission from an excited state to a lower state. By adjusting the delay in time between the k12-excitation and the subsequent radiation field (hereafter referred to as tdelay) different stages of relaxation following the k12-excitation can be specifically manipulated and interrogated by the subsequent radiation field. Following k12-excitation, relaxation takes place, by vibrational relaxation within the electronic state of S1 (typically ps range), by electronic relaxation of S1 to S0 (typically ns range), and by relaxation into more long-lived transient states (typically μs range and longer). Following k12-excitation, the extent of vibrational relaxation at the time the subsequent radiation field is applied (at tdelay) influences the energy differences between S1 and its neighbouring states, and thereby the spectral dependence for excitation or stimulated emission taking place from S1 by the subsequent radiation field. On a longer time scale (typically ns), the extent of electronic relaxation at tdelay determines the fraction of fluorescent molecules that still are in S1 that can be excited to higher excited states or that can be subject to stimulated emission to a lower state. Similarly, at yet longer time scales, the extent relaxation to long-lived transient states has taken place at tdelay determines what transitions that can be generated by the subsequent radiation field, simply by the distribution of states being present at a certain tdelay after onset of the excitation radiation field (e.g. 1406 in FIG. 14A), determining the possible “starting points” for possible transitions influenced by this subsequent radiation field.
Fluorescence quenching experiments normally measures the extent of excited state depopulation as a consequence of fluorophore-quencher interaction. Since the fluorescence lifetime typically is in the nanosecond time range, the time available for interaction is about 1000 times shorter than for long-lived states, like triplet states. Procedures for triplet state quenching measurements has indeed also been implemented. However, a problem then is the monitoring of the triplet state population. It is usually monitored via transient triplet state absorption or via phosphorescence, generated by decay of the triplet state to the ground singlet state, but this is normally technically complicated to do and insensitive.
The FCS approach for triplet state monitoring relies on a strong excitation of the fluorescent molecules, such that the response in fluorescence intensity per molecule to the excitation intensity (or irradiance, in W/cm2) is no longer linear in the excitation intensity range used, i.e. that the mean probability of populating the excited singlet state S1 is no longer proportional to the excitation intensity. In FCS measurements high enough excitation intensities can easily be reached due to a strong focussing of the excitation laser beam (e.g. 1406 in FIG. 14A) under the microscope objective, where the molecules to be investigated are located. In fact, focussing the laser beam at or close to the limit of diffraction, with a beam waist radius in the focus of approximately 0.3 μm, excitation powers of less than 1 mW are sufficient to yield prominent triplet state populations in the fluorophores within the laser beam focal volume. Moreover, due to the small size of the focus, freely moving molecules typically pass the laser beam focal volume within times short enough to avoid photobleaching. Alternatively, given a laser focus for excitation, the passage times of the molecules, i.e. the exposure time to the excitation laser (e.g. 1406 in FIG. 14A), can be made shorter by applying either a scan or flow of the sample (with a stationary beam), or scan the beam (with a stationary sample).
Apart from molecular monitoring via triplet state quenching/induction (as outlined above), triplet state parameters can be exploited as a readout for intra- and intermolecular distances via so-called Fluorescence (Förster) Resonance Energy Transfer (FRET) FRET is a photo-physical process, and a well established tool to yield molecular distance information in the range of 10-100 Å (see T Förster, Ann. Phys. 1948, 2: p 55; L Stryer, R P Haugland, Proc. Natl. Acad. Sci, 1967, 58: p. 719) Via FRET, energy is transferred non-radiatively by resonance from a donor molecule to an acceptor molecule via an induced dipole-induced dipole interaction. The efficiency, E, with which transfer of excitation energy takes place depends on several parameters, such as the mutual orientation of the dipole moments of the fluorophores, the spectral overlap etc.
However, the usefulness of FRET relies primarily on the fact that E depends on the inverse-sixth-power of the distance, RDA, between the donor and acceptor dye molecules, FRET thus provides what is referred to as a “molecular ruler”. In traditional fluorescence spectroscopy, the FRET efficiency E can be monitored via several parameters, most commonly via the intensities of the donor and acceptor fluorophores (donor fluorescence decreases and acceptor fluorescence increases with higher E), or the lifetime of the donor (higher E leads to shorter donor fluorescence lifetimes). FRET has found a wide range of applications in the biomedical field. By use of microscopic techniques it has been possible to apply FRET to many different microenvironments, with little or no interference of the system under study FRET has also found a wide application as a basis for molecular binding assays. A photophysical framework for fluorescence signals generated by FRET being sensitive to molecular conformation, association and separation is disclosed in the article by BE A, Jares-Erdman and T. M. Jovin, Nature Biotechnology, Vol. 21, No. 11, November 2003.
Irrespective via what parameters the FRET efficiency is read-out, also this method has certain disadvantages. In particular, it can be problematic to account for incomplete labelling of the molecules, such that the calculation of E is based on a collection of molecules, containing a substantial fraction of molecules not labelled with both donor and acceptor fluorophores. Cross-talk of fluorescence can also cause problems, such that fluorescence of the donor is erroneously partly detected in the detector for the acceptor fluorescence, and vice versa.
Probing of triplet state properties by the proposed method is also likely to be useful for High-Throughput Screening (HTS), where molecular interactions are investigated in a highly parallelised and fast manner. Often, bioactive molecules are only available in minute amounts, but need to be discriminated against, and checked in terms of their interaction with, a large number of compounds. Because of its high sensitivity (single-molecule detection), fast read-out, and high specificity (many parameters available), fluorescence spectroscopy has become a key technology in biotechnology for drug discovery and diagnostics. As an indication of the commercial importance, one can refer to the very strong interest from pharmaceutical companies from all over the world to use ultra-sensitive fluorescence techniques for high-throughput screening of new potential drugs. Many of the HTS assays developed, involves FRET (and its inherent problems).